Transitional B cells commit to marginal zone B cell fate by Taok3-mediated surface expression of ADAM10.

Notch2 and B cell antigen receptor (BCR) signaling determine whether transitional B cells become marginal zone B (MZB) or follicular B (FoB) cells in the spleen, but it is unknown how these pathways are related. We generated Taok3-/- mice, lacking the serine/threonine kinase Taok3, and found cell-intrinsic defects in the development of MZB but not FoB cells. Type 1 transitional (T1) B cells required Taok3 to rapidly respond to ligation by the Notch ligand Delta-like 1. BCR ligation by endogenous or exogenous ligands induced the surface expression of the metalloproteinase ADAM10 on T1 B cells in a Taok3-dependent manner. T1 B cells expressing surface ADAM10 were committed to becoming MZB cells in vivo, whereas T1 B cells lacking expression of ADAM10 were not. Thus, during positive selection in the spleen, BCR signaling causes immature T1 B cells to become receptive to Notch ligands via Taok3-mediated surface expression of ADAM10.

S. mansoni -derived omega-1 prevents OVA-specific allergic airway inflammation via hampering of cDC2 migration.

Chronic infection with Schistosoma mansoni parasites is associated with reduced allergic sensitization in humans, while schistosome eggs protects against allergic airway inflammation (AAI) in mice. One of the main secretory/excretory molecules from schistosome eggs is the glycosylated T2-RNAse Omega-1 (ω1). We hypothesized that ω1 induces protection against AAI during infection. Peritoneal administration of ω1 prior to sensitization with Ovalbumin (OVA) reduced airway eosinophilia and pathology, and OVA-specific Th2 responses upon challenge, independent from changes in regulatory T cells. ω1 was taken up by monocyte-derived dendritic cells, mannose receptor (CD206)-positive conventional type 2 dendritic cells (CD206+ cDC2), and by recruited peritoneal macrophages. Additionally, ω1 impaired CCR7, F-actin, and costimulatory molecule expression on myeloid cells and cDC2 migration in and ex vivo, as evidenced by reduced OVA+ CD206+ cDC2 in the draining mediastinal lymph nodes (medLn) and retainment in the peritoneal cavity, while antigen processing and presentation in cDC2 were not affected by ω1 treatment. Importantly, RNAse mutant ω1 was unable to reduce AAI or affect DC migration, indicating that ω1 effects are dependent on its RNAse activity. Altogether, ω1 hampers migration of OVA+ cDC2 to the draining medLn in mice, elucidating how ω1 prevents allergic airway inflammation in the OVA/alum mouse model.

IRF8 Transcription Factor Controls Survival and Function of Terminally Differentiated Conventional and Plasmacytoid Dendritic Cells, Respectively.

Interferon regulatory factor-8 (IRF8) has been proposed to be essential for development of monocytes, plasmacytoid dendritic cells (pDCs) and type 1 conventional dendritic cells (cDC1s) and remains highly expressed in differentiated DCs. Transcription factors that are required to maintain the identity of terminally differentiated cells are designated "terminal selectors." Using BM chimeras, conditional Irf8(fl/fl) mice and various promotors to target Cre recombinase to different stages of monocyte and DC development, we have identified IRF8 as a terminal selector of the cDC1 lineage controlling survival. In monocytes, IRF8 was necessary during early but not late development. Complete or late deletion of IRF8 had no effect on pDC development or survival but altered their phenotype and gene-expression profile leading to increased T cell stimulatory function but decreased type 1 interferon production. Thus, IRF8 differentially controls the survival and function of terminally differentiated monocytes, cDC1s, and pDCs.

The STE20 kinase TAOK3 controls the development of house dust mite-induced asthma in mice.

BACKGROUND: The most common endotype of asthma is type 2-high asthma, which is sometimes driven by adaptive allergen-specific TH2 lymphocytes that react to allergens presented by dendritic cells (DCs), or sometimes by an innate immune response dominated by type 2 innate lymphocytes (ILC2s). Understanding the underlying pathophysiology of asthma is essential to improve patient-tailored therapy. The STE20 kinase thousand-and-one kinase 3 (TAOK3) controls key features in the biology of DCs and lymphocytes, but to our knowledge, its potential usefulness as a target for asthma therapy has not yet been addressed. OBJECTIVE: We examined if and how loss of Taok3 affects the development of house dust mite (HDM)-driven allergic asthma in an in vivo mouse model. METHODS: Wild-type Taok3+/+ and gene-deficient Taok3-/- mice were sensitized and challenged with HDM, and bronchoalveolar lavage fluid composition, mediastinal lymph node cytokine production, lung histology, and bronchial hyperreactivity measured. Conditional Taok3fl/fl mice were crossed to tissue- and cell-specific specific deletor Cre mice to understand how Taok3 acted on asthma susceptibility. Kinase-dead (KD) Taok3KD mice were generated to probe for the druggability of this pathway. Activation of HDM-specific T cells was measured in adoptively transferred HDM-specific T-cell receptor-transgenic CD4+ T cells. ILC2 biology was assessed by in vivo and in vitro IL-33 stimulation assays in Taok3-/- and Taok3+/+, Taok3KD, and Red5-Cre Taok3fl/fl mice. RESULTS: Taok3-/- mice failed to mount salient features of asthma, including airway eosinophilia, TH2 cytokine production, IgE secretion, airway goblet cell metaplasia, and bronchial hyperreactivity compared to controls. This was due to intrinsic loss of Taok3 in hematopoietic and not epithelial cells. Loss of Taok3 resulted in hampered HDM-induced lung DC migration to the draining lymph nodes and defective priming of HDM-specific TH2 cells. Strikingly, HDM and IL-33-induced ILC2 proliferation and function were also severely affected in Taok3-deficient and Taok3KD mice. CONCLUSIONS: Absence of Taok3 or loss of its kinase activity protects from HDM-driven allergic asthma as a result of defects in both adaptive DC-mediated TH2 activation and innate ILC2 function. This identifies Taok3 as an interesting drug target, justifying further testing as a new treatment for type 2-high asthma.

The ORMDL3 asthma susceptibility gene regulates systemic ceramide levels without altering key asthma features in mice.

BACKGROUND: Genome-wide association studies in asthma have repeatedly identified single nucleotide polymorphisms in the ORM (yeast)-like protein isoform 3 (ORMDL3) gene across different populations. Although the ORM homologues in yeast are well-known inhibitors of sphingolipid synthesis, it is still unclear whether and how mammalian ORMDL3 regulates sphingolipid metabolism and whether altered sphingolipid synthesis would be causally related to asthma risk. OBJECTIVE: We sought to examine the in vivo role of ORMDL3 in sphingolipid metabolism and allergic asthma. METHODS: Ormdl3-LacZ reporter mice, gene-deficient Ormdl3-/- mice, and overexpressing Ormdl3Tg/wt mice were exposed to physiologically relevant aeroallergens, such as house dust mite (HDM) or Alternaria alternata, to induce experimental asthma. Mass spectrometry-based sphingolipidomics were performed, and airway eosinophilia, TH2 cytokine production, immunoglobulin synthesis, airway remodeling, and bronchial hyperreactivity were measured. RESULTS: HDM challenge significantly increased levels of total sphingolipids in the lungs of HDM-sensitized mice compared with those in control mice. In Ormdl3Tg/wt mice the allergen-induced increase in lung ceramide levels was significantly reduced, whereas total sphingolipid levels were not affected. Conversely, in liver and serum, levels of total sphingolipids, including ceramides, were increased in Ormdl3-/- mice, whereas they were decreased in Ormdl3Tg/wt mice. This difference was independent of allergen exposure. Despite these changes, all features of asthma were identical between wild-type, Ormdl3Tg/wt, and Ormdl3-/- mice across several models of experimental asthma. CONCLUSION: ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3 expression does not result in altered experimental asthma.

IL-33trap is a novel IL-33-neutralizing biologic that inhibits allergic airway inflammation.

BACKGROUND: The emergence of IL-33 as a key molecular player in the development and propagation of widespread inflammatory diseases, including asthma and atopic dermatitis, has established the need for effective IL-33-neutralizing biologics. OBJECTIVE: Here we describe the development and validation of a new antagonist of IL-33, termed IL-33trap, which combines the extracellular domains of the IL-33 receptor (ST2) and its coreceptor, IL-1 receptor accessory protein, into a single fusion protein. METHODS: We produced and purified recombinant IL-33trap from human cells and analyzed its IL-33-binding affinity and IL-33 antagonistic activity in cultured cells and mice. IL-33trap activity was also benchmarked with a recombinant soluble ST2 corresponding to the naturally occurring IL-33 decoy receptor. Finally, we studied the effect of IL-33trap in the Alternaria alternata mouse model of allergic airway inflammation. RESULTS: In vitro IL-33trap binds IL-33 and inhibits IL-33 activity to a much stronger degree than soluble ST2. Furthermore, IL-33trap inhibits eosinophil infiltration, splenomegaly, and production of signature cytokines in splenic lymphocytes and lung tissue on IL-33 injection. Finally, administration of IL-33trap at the time of allergen challenge inhibits inflammatory responses in a preclinical mouse model of acute allergic airway inflammation. CONCLUSIONS: IL-33trap is a novel IL-33 antagonist that outperforms the natural IL-33 decoy receptor and shows anti-inflammatory activities in a preclinical mouse model of acute allergic airway inflammation when administered at the time of allergen challenge.

A bispecific antibody strategy to target multiple type 2 cytokines in asthma.

BACKGROUND: Asthma is a chronic inflammatory airway disease in which innate and adaptive immune cells act together to cause eosinophilic inflammation, goblet cell metaplasia (GCM), and bronchial hyperreactivity (BHR). In clinical trials using biologicals against IL-4 receptor (IL-4R) α or IL-5, only a subset of patients with moderate-to-severe asthma responded favorably, suggesting that distinct pathophysiologic mechanisms are at play in subgroups of patients called endotypes. However, the effect of multiple cytokine blockade using bispecific antibodies has not been tested. OBJECTIVE: We sought to target simultaneously the IL-4, IL-13, and IL-5 signaling pathways with a novel IL-4Rα/IL-5-bispecific antibody in a murine house dust mite (HDM) model of asthma. METHODS: Two mAbs neutralizing IL-4Rα and IL-5 were generated by using a llama-based antibody platform. Their heavy and light chains were then cotransfected in mammalian cells, resulting in a heterogeneous antibody mixture from which the bispecific antibody was isolated by using a dual anti-idiotypic purification process. C57BL/6J mice were finally sensitized and challenged to HDM extracts and treated during challenge with the antibodies. RESULTS: We successfully generated and characterized the monospecific and bispecific antibodies targeting IL-4Rα and IL-5. The monospecific antibodies could suppress eosinophilia, IgE synthesis, or both, whereas only the IL-4Rα/IL-5-bispecific antibody and the combination of monospecific antibodies additionally inhibited GCM and BHR. CONCLUSION: Type 2 cytokines act synergistically to cause GCM and BHR in HDM-exposed mice. These preclinical results show the feasibility of generating bispecific antibodies that target multiple cytokine signaling pathways as superior inhibitors of asthma features, including the difficult-to-treat GCM.

Lung tumours reprogram pulmonary dendritic cell immunogenicity at the microRNA level.

Lung cancer arises in a context of tumour-induced immune suppression. Dendritic cells (DCs) are central players in the induction of anti-tumoural immunity, providing critical signals that drive the induction of cytotoxic T-cell responses. Meanwhile, microRNAs are associated with tumour development as well as immune regulation. We postulated that lung tumours escape immune control by reprogramming DC immunogenicity at the microRNA level. Using an orthotopic model of lung cancer, we first identified the DC population responsible for transport and cross-presentation of lung tumour-derived antigens to naïve T cells in the draining mediastinal lymph nodes (LNs). Profiling the full microRNA repertoire of these DCs revealed a restricted set of microRNAs that was consistently dysregulated in the presence of lung tumours, with miR-301a as one of the top upregulated transcripts. Overexpression of miR-301a in DCs suppressed IL-12 secretion, decreased IFN-γ release from antigen-specific cytotoxic T cells, and shifted antigen-specific T helper cytokine profile away from IFN-γ towards IL-13 and IL-17A-secreting T cells. Strikingly, DC-selective Dicer1 gene deletion resulted in delayed lung tumour growth and a survival benefit. Taken together, our data reveal that lung tumours induce an immunosuppressive microRNA signature in pulmonary DCs. Interfering with the DC-intrinsic capacity to remodel microRNA repertoires affects lung tumour outcome.

Canonical IRE1 function needed to sustain vigorous natural killer cell proliferation during viral infection.

The unfolded protein response (UPR) aims to restore ER homeostasis under conditions of high protein folding load, a function primarily serving secretory cells. Additional, non-canonical UPR functions have recently been unraveled in immune cells. We addressed the function of the inositol-requiring enzyme 1 (IRE1) signaling branch of the UPR in NK cells in homeostasis and microbial challenge. Cell-intrinsic compound deficiency of IRE1 and its downstream transcription factor XBP1 in NKp46+ NK cells, did not affect basal NK cell homeostasis, or overall outcome of viral MCMV infection. However, mixed bone marrow chimeras revealed a competitive advantage in the proliferation of IRE1-sufficient Ly49H+ NK cells after viral infection. CITE-Seq analysis confirmed strong induction of IRE1 early upon infection, concomitant with the activation of a canonical UPR signature. Therefore, we conclude that IRE1/XBP1 activation is required during vigorous NK cell proliferation early upon viral infection, as part of a canonical UPR response.

The ubiquitin-editing enzyme A20 controls NK cell homeostasis through regulation of mTOR activity and TNF.

The ubiquitin-editing enzyme A20 is a well-known regulator of immune cell function and homeostasis. In addition, A20 protects cells from death in an ill-defined manner. While most studies focus on its role in the TNF-receptor complex, we here identify a novel component in the A20-mediated decision between life and death. Loss of A20 in NK cells led to spontaneous NK cell death and severe NK cell lymphopenia. The few remaining NK cells showed an immature, hyperactivated phenotype, hallmarked by the basal release of cytokines and cytotoxic molecules. NK-A20-/- cells were hypersensitive to TNF-induced cell death and could be rescued, at least partially, by a combined deficiency with TNF. Unexpectedly, rapamycin, a well-established inhibitor of mTOR, also strongly protected NK-A20-/- cells from death, and further studies revealed that A20 restricts mTOR activation in NK cells. This study therefore maps A20 as a crucial regulator of mTOR signaling and underscores the need for a tightly balanced mTOR pathway in NK cell homeostasis.

Regulated IRE1-dependent mRNA decay sets the threshold for dendritic cell survival.

The IRE1-XBP1 signalling pathway is part of a cellular programme that protects against endoplasmic reticulum (ER) stress, but also controls development and survival of immune cells. Loss of XBP1 in splenic type 1 conventional dendritic cells (cDC1s) results in functional alterations without affecting cell survival. However, in mucosal cDC1s, loss of XBP1 impaired survival in a tissue-specific manner-while lung cDC1s die, intestinal cDC1s survive. This was not caused by differential activation of ER stress cell-death regulators CHOP or JNK. Rather, survival of intestinal cDC1s was associated with their ability to shut down protein synthesis through a protective integrated stress response and their marked increase in regulated IRE1-dependent messenger RNA decay. Furthermore, loss of IRE1 endonuclease on top of XBP1 led to cDC1 loss in the intestine. Thus, mucosal DCs differentially mount ATF4- and IRE1-dependent adaptive mechanisms to survive in the face of ER stress.

Cholesterol-sensing liver X receptors stimulate Th2-driven allergic eosinophilic asthma in mice.

INTRODUCTION: Liver X receptors (LXRs) are nuclear receptors that function as cholesterol sensors and regulate cholesterol homeostasis. High cholesterol has been recognized as a risk factor in asthma; however, the mechanism of this linkage is not known. METHODS: To explore the importance of cholesterol homeostasis for asthma, we investigated the contribution of LXR activity in an ovalbumin- and a house dust mite-driven eosinophilic asthma mouse model. RESULTS: In both models, airway inflammation, airway hyper-reactivity, and goblet cell hyperplasia were reduced in mice deficient for both LXRα and LXRß isoforms (LXRα(-/-)ß(-/-)) as compared to wild-type mice. Inversely, treatment with the LXR agonist GW3965 showed increased eosinophilic airway inflammation. LXR activity contributed to airway inflammation through promotion of type 2 cytokine production as LXRα(-/-)ß(-/-) mice showed strongly reduced protein levels of IL-5 and IL-13 in the lungs as well as reduced expression of these cytokines by CD4(+) lung cells and lung-draining lymph node cells. In line herewith, LXR activation resulted in increased type 2 cytokine production by the lung-draining lymph node cells. CONCLUSIONS: In conclusion, our study demonstrates that the cholesterol regulator LXR acts as a positive regulator of eosinophilic asthma in mice, contributing to airway inflammation through regulation of type 2 cytokine production.

Farm dust and endotoxin protect against allergy through A20 induction in lung epithelial cells.

Growing up on a dairy farm protects children from allergy, hay fever, and asthma. A mechanism linking exposure to this endotoxin (bacterial lipopolysaccharide)-rich environment with protection has remained elusive. Here we show that chronic exposure to low-dose endotoxin or farm dust protects mice from developing house dust mite (HDM)-induced asthma. Endotoxin reduced epithelial cell cytokines that activate dendritic cells (DCs), thus suppressing type 2 immunity to HDMs. Loss of the ubiquitin-modifying enzyme A20 in lung epithelium abolished the protective effect. A single-nucleotide polymorphism in the gene encoding A20 was associated with allergy and asthma risk in children growing up on farms. Thus, the farming environment protects from allergy by modifying the communication between barrier epithelial cells and DCs through A20 induction.